POS0225 FLUIDIC SHEAR STRESS REDUCES TNFΑ-MEDIATED CARTILAGE DAMAGE IN A 3D MODEL OF DEGENERATIVE JOINT DISEASE
Authors: D. H. Do Nguyen, C. Lubahn, T. Leeuw, F. Buttgereit, T. Gaber, A. Damerau
Journal: Annals of the Rheumatic Diseases (2022)

Background Pathomechanisms of degenerative joint diseases such as osteoarthritis (OA) ultimately result in the breakdown of cartilage tissue. To date, the exact underlying mechanisms of both cause and progression of OA remain unclear. Therefore, developing complex and long-lasting in vitro components of a human joint including cartilage, subchondral bone, synovial membrane and tendons that simulate the 3D architecture and the metabolic, humoral and cellular interplay of the joint components is needed to study the long-lasting course of OA pathogenesis. Beside the impact of metabolic components and 3D architecture, mechanical forces are well-known to be important modulators of joint health, while aberrant forces are primary etiological factors leading to cartilage degeneration. Objectives Here, we aimed to (i) develop a long-lasting human in vitro 3D cartilage model using alternated perfused cultivation and (ii) simulate TNFα-mediated cartilage degradation. As a mechanical force we used the perfusion-mediated fluid shear stress (FSS) to enhance chondrogenesis and mimic FSS during joint movement. Methods Human bone marrow-derived mesenchymal stromal cells (MSC) were used to develop an in vitro 3D cartilage model incubated in a bioreactor with a perfusion cycle that facilitates mechanical stimulation via FSS and daily sampling. Within the bioreactor, MSC mass cultures were subjected to FSS at 10 dyn/cm 2 by medium circulation three times a day for 1.5 hours. The approach of using optimized FSS rate, cycles and cultivation period of 18 days for MSC mass cultures was compared to a non-perfused control based on cell viability (live-dead- and viability-assay), apoptosis (TUNEL-assay, caspase-3/7-activity, BCL2/BAX ), metabolic activity (oxygen and glucose consumption, lactate production), chondrogenic gene expression ( ACAN, COMP , COL2A1, COL1A1 , COL2A1/COL1A1 ) and matrix metalloproteinase expression (MMP-1, -3, -13 ). Results Alternate perfused long-term cultivation at 10 dyn/cm 2 did not affect cell survival; it rather reduced apoptosis, did not affect oxygen consumption but reduced glucose consumption and lactate production and enhanced chondrogenic gene expression with reduced MMP13 and COMP gene expression compared with non-perfused conditions. Mimicking pathophysiology of OA we stimulated the 3D cartilage model with 100 ng/mL TNFα for 6 hours under non-perfused and perfused long-term cultivation with FSS at 10 dyn/cm 2 as a mechanical stimulus. Compared to untreated perfused conditions, TNFα stimulation (i) did not affect overall cell survival but enhanced apoptosis (demonstrating efficacy of stimulation), (ii) did not affect oxygen consumption and glycolysis, and (iii) enhanced COMP and MMP1 expression as markers of matrix protein turnover. In comparison to TNFα treated cells under non-perfused conditions, TNF stimulation under perfused conditions (i) did not affect cell survival but reduced apoptosis, (ii) did not affect oxygen consumption but reduced glucose consumption and lactate production as a measure of glycolysis, and (iii) reduced the expression of IL6 and soluble amounts of IL-6 but not of TNFA whereas soluble amounts of TNFα were enhanced. Furthermore, TNFα stimulation (iv) reduced the expression of matrix degrading enzymes but (v) enhanced anabolic chondrogenic matrix proteins on mRNA. Conclusion In a 3D model that mimics OA, FSS as a mechanical stimulus provides a metabolic “feel-good” niche that reduces chondrocyte apoptosis, metabolic activity, and matrix metalloproteinase expression, increases matrix protein expression and protects against TNFα-mediated cartilage degradation. Acknowledgements This project is funded by Sanofi-Aventis Deutschland GmbH. Disclosure of Interests Duc Ha Do Nguyen: None declared, Christina Lubahn: None declared, Thomas Leeuw Employee of: Thomas Leeuw is a Sanofi employee and may hold shares and/or stock options in the company., Frank Buttgereit: None declared, Timo Gaber: None declared, Alexandra Damerau: None declared